Astrocytes, the most abundant glial cells in the mammalian central nervous system (CNS), exhibit a special form of excitability (cytoplasmic excitability), characterized by elevations in the cytosolic free Ca2+ concentration ([Ca2+]i), which are elicited by various transmitters and chemical messengers and affects many important cellular processes, including the exocytotic release of gliotransmitters, such as glutamate, ATP and peptides. In addition to Ca2+ as a second messenger, cyclic adenosine monophosphate (cAMP) modulates a large variety of cellular functions and regulates numerous biological processes in the brain and in astrocytes. cAMP synthesis is mainly triggered by agonist-induced activation of transmembrane G protein-coupled receptors (GPCRs) and subsequent activation of adenylyl cyclases (AC) at the inner site of the plasma membrane. cAMP activates a limited number of effectors in the cell, primarily the cAMP-dependent protein kinase (PKA) which, by phosphorylating cytoplasmic and nuclear targets mediates many different functional effects, although signaling via cAMP-activated GTP-exchange protein Epac, and via cAMP-gated ion channels is also present. The cellular content of cAMP is tightly controlled by GPCRs via both ACs and cAMP-degrading phosphodiesterases (PDEs).
Astrocytes express several types of GPCRs (e.g. β-adrenergic receptors (β-AR), lactate receptors, metabotropic glutamate receptors, adenosine receptors, and others). β-ARs are abundantly present on astrocytes in both white and grey matter of the brain and regulate important astrocyte functions via activation/inhibition of cAMP dependent pathways. The activation of β-AR/cAMP signaling pathway in astrocytes by the “fight or flight response” neurotransmitter/hormone noradrenaline/adrenaline (NA/ADR), respectively, has been shown to promote rapid degradation of glycogen in astrocytes, which serves as the main brain energy reserve. In addition, NA may also elevate cytosolic glucose (Prebil et al., 2011) and glucose uptake via β-AR/cAMP signaling (Prebil et al., 2011) and increase glycogen content (Allaman et al., 2003). β-AR stimulation can induce the expression of cytokine IL-6 in astrocytes and neurotrophic factors, it can modulate glial inwardly rectifying potassium channels Kir, extracellular concentration of adenosine, and glutamate.
Impaired regulation of astrocytic β2-AR/cAMP pathway is considered to contribute to the pathophysiology of several neurological conditions such as multiple sclerosis (Laureys et al., 2010) and Alzheimer's disease (Lee et al., 1997). Astroglial β-ARs are also functionally regulating astrocyte cellular morphology (Hatton et al., 1991). An increase in intracellular cAMP production upon β-AR stimulation induces astrocyte stellation, transformation from a flattened irregular morphology to a stellate, process-bearing morphology (Bicknell et al., 1989; Shain et al., 1987).
Lactate is considered to have two roles in the brain. It is a fuel and also likely acts on the plasma receptor GPR81 (Bergersen and Gjedde, 2012), originally discovered in adipose tissue, where GPR81 is highly expressed and serves to down-regulate the formation of cAMP, thereby curbing lipolysis and promoting energy storage. Interestingly, the neuroprotective role of lactate in the brain has been considered in ischemic, excitotoxic and mechanical insults (Cureton et al., 2010; Ros et al., 2001; Schurr et al., 2001). These effects are not easily explained solely by the role of lactate as a fuel, but indicate that lactate plays also a role in signalling, likely via the GPR81 receptor. However, direct real-time measurements of activation of this receptor in astrocytes have not been conducted. The results in this study show that GPR81 is present in astrocytes and that the activation of this receptor by lactate or 3-Chloro-5-hydroxybenzoic acid (3-Cl-5-HBA), an agonist of this receptor (Ahmed et al., 2009), elevates cytosolic cAMP and consequently also cytosolic glucose (Prebil et al., 2011).
The real-time dynamics of β-AR and GPR81 mediated cAMP signaling in live single astrocyte have not been reported. It is also unclear how the activation of β-ARs affects astrocyte morphology (cell area and perimeter). Genetically encoded FRET biosensors that enable direct monitoring of rapid changes in free cytosolic cAMP were developed recently. These sensors are based on downstream cAMP targets, including cAMP-dependent PKA, cAMP-gated ion channels, and cAMP-activated GTP-exchange protein Epac.
The cAMP level has never been used as a target for substances useful in the treatment of the pathophysiological states, such as CNS trauma, cognitive deficits, autism, neuroinflammation, epilepsy, neuroprotection, and neurodegenerative disorders, e.g. multiple sclerosis, Alzheimer's disease.
Central to the hypothesis of the tripartite synapse involves astrocyte cytoplasmic excitability. However, the knowledge of how this is attenuated is fragmental, especially in pathophysiological conditions. The response to traumatic CNS injury involves astroglial edema and likely also modifications in cytoplasmic excitability of cells. In cultured astrocytes hypotonic stimulation causes swelling and morphological changes due to membrane unfolding, not vesicle fusion (Pangrsic et al. 2006). Elevations in intracellular cAMP levels via purinergic and adenosine receptors were linked to reduced hypotonic swelling of retinal glial cells (Wurm et al., 2009). However, the involvement of cAMP signaling in astrocyte swelling has not been studied yet.
Against the background, it is an object of the present invention to provide targets for substances useful in the treatment of specific diseases, in particular of astroglial edema resulting from CNS trauma, cognitive deficit, autism, neuroinflammation, epilepsy and neurodegenerative disorders, such as multiple sclerosis, Alzheimer's disease.